1. Field of the Invention
The present invention relates to a novel method for culturing multi-layered tissues in vitro, wherein tissues and cells, by taking advantage of their disparity in volume, are to be distributed into different layers of a specific multi-layer porous carrier. When cultured in vitro, the tissues and cells grown in different layers of said carrier are to reconstruct a multi-layer tissue, so as to overcome the current technical bottleneck caused by the unavailability in culturing multi-layered tissues in vitro.
2. Description of the Prior Art
Due to the anticipated arrival of an aged society and an increasing number of people suffering from work or sports-related injuries, pathological changes on articular cartilage that causes painful reactions are clinical symptoms that cannot be overlooked. The articular cartilage lesions are classified into two groups according to their severity: partial thickness defect and full thickness defect. Partial thickness defect is the injury or erosion on the cartilage tissue of articular surface that does not reach the subchondral bone. Full thickness defect is the injury or erosion on the cartilage tissue that penetrates the subchondral bone. With the advancement in surgery operation and arthroscopy, partial thickness defect of articular cartilage lesions can be treated or its symptom can be relieved by surgery and arthroscopic methods such as abrasion arthroplasty, debridement and lavage, high tibial osteotomy, microfracturing, and drilling. However, general arthroscopy cannot cure full thickness defect where the damaged area is much wider and/or deeper than that of partial thickness defect. As a result, patients are faced with the only choice of undergoing both joint excision and replacement with an artificial joint to relieve pain and regain function of joints. It is estimated that over 150,000 knee replacement operations caused by full thickness defect are performed annually in the U.S., and the number of such operations is rising year after year. Because artificial joints are expensive, medical costs for hospitalization and surgery are high. Furthermore, because artificial joints are made of metals that last only about 10 to 20 years after being transplanted into human bodies, young patients must suffer from the pain of going through another surgical procedure in the future, while older patients, who, more often than not, are unable to go through another surgery, become handicapped and walking impaired, placing heavy burdens on both families and society. Therefore, there is a great need for the development of a technique for treating full-thickness defects.
In 1995, a Hungarian surgeon developed a technique for autologous implantation called Mosaic Plasty Procedure. Relying on arthroscopy, Mosaic Plasty Procedure is performed with a tube chisel that harvests a cylindrical plug of healthy cartilage and its subjacent subchondral bone from the patient's non-weight-bearing surface at the joints. Next, the damaged site is drilled with a tube chisel to make a hole of the same diameter as that of the cylindrical plug. Then, the cylindrical plug previously harvested from healthy cartilage is to be inserted into the hole of the damaged site. The procedures may be repeated several times when the damaged area is wide. By performing this technique, a new articular surface at damage site is formed. Because the mosaic appearance of new articular surface after surgery, the technique is termed Mosaic Plasty Procedure. Such technique has the merits of using patients' own tissues for transplantation, and thereby avoids immunogenic problems caused by using allograft or xenograft. Furthermore, the cylindrical plug is composed of biphasic joint, which contains cartilage and subjacent subchondral bone. After transplantation, peripheral bone tissue will grow into the spongy bone tissue of subchondral bone at the site of lesion. Such growth contributes to fixation of the transplant and reduces the problems caused by either suture fixation or transplant loosening. However, the autologous cartilage for transplantation is usually harvested from the non-weight-bearing surface of patients' joints, where the available area and volume are limited, and hence treating larger damaged area becomes unfeasible. Accordingly, the bottleneck in current technique is in the development of in vitro culturing technology to amplify the volume of autologous biphasic joint while maintaining the original characteristics of cartilage tissue.
The tissue engineering technique nowadays is limited to culturing single tissue, while in clinical application, tissues to be filled or restored are often composite tissues. For example, the human articular surface is a typical biphasic composite tissue. Current tissue engineering-related technique is mainly to utilize patients' own cells in cooperation with different porous carriers to reconstruct, in vitro or in vivo, the original framework of tissues by taking advantage of the three-dimensional structures of porous carriers. Generally speaking, such technique can only be used for culturing homogenous single tissues but not multi-layer tissues. Because the diameter of cells is smaller than that of the pore of porous carrier, when seeding two different cells in porous carrier, cells would flow around the carrier and mix-up but fail to grow in an orderly pattern into a multi-layer tissue. The present related technique is to grow and proliferate chondrocytes and osteocytes separately in vitro, followed by seeding cells so as to attach and incubate within two individual porous substrates to grow into tissues, and finally combine two tissues to obtain a multi-layer tissue by fusing their interfaces. This approach is time-consuming and a more practical technique is still under development.